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Douglas Instruments Microseeding slide 6 Protein crystallization Step 1: screening with random solutions that have given crystals before x 96 Step 2: optimization by making small changes Modify your protein or make a new construct

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Douglas Instruments Microseeding slide 7 Protein crystallization Step 1: screening with random solutions that have given crystals before x 96 Step 1.5: random microseeding Step 2: optimization by making small changes Modify your protein or make a new construct

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Douglas Instruments Microseeding slide 8 Protein crystallization Step 1: screening with random solutions that have given crystals before x 96 Step 1.5: random microseeding Step 2: optimization by making small changes Modify your protein or make a new construct

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Douglas Instruments Microseeding slide 41 See www.douglas.co.uk/mms.com or sheetwww.douglas.co.uk/mms.com 1.Break crystals with a probe 2.Place contents of well in 50 μl of reservoir solution 3.Vortex with Hampton “Seed Bead” 4.Make a dilution series immediately 5.Freeze Look after your seeds! How to make the seed stock

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Douglas Instruments Microseeding slide 43 Membrane proteins Christine Oswald (Goethe University of Frankfurt) pointed out that the seeds may dissolve if there is not enough detergent We recommend crushing the crystals and harvesting several large drops without dilution Only 1.5 µl of seed stock are needed to fill a whole plate with (the right kind of) contact dispenser See http://www.douglas.co.uk/MMS_proc.htmhttp://www.douglas.co.uk/MMS_proc.htm

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Douglas Instruments Microseeding slide 52 Microseeding Opticryst – a consortium of European institutions and companies aiming to improve crystal optimization. 2007 – 2010. Stefan set up 30,000 drops and estimated the number of crystals In 15,000 drops!

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Douglas Instruments Microseeding slide 53 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? (2) How stable are the seed stocks? (3) Is “preseeding” the protein stock helpful? (4) How can we avoid salt crystals? (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

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Douglas Instruments Microseeding slide 55 “Receptive” conditions Conditions where: (1)crystals don’t grow without seeds in four drops, but (2)crystals grow in at least three out of four drops with seeds.

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Douglas Instruments Microseeding slide 56 “Receptive” conditions Conditions where: (1)crystals don’t grow without seeds in four drops, but (2)crystals grow in at least three out of four drops with seeds. 25 receptive conditions were found

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Douglas Instruments Microseeding slide 61 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? (3) Is “preseeding” the protein stock helpful? (4) How can we avoid salt crystals? (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

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Douglas Instruments Microseeding slide 64 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful? (4) How can we avoid salt crystals? (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

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Douglas Instruments Microseeding slide 65 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Please read the paper! (4) How can we avoid salt crystals?Please read the paper! (5) How can we get more diverse crystals?Please read the paper! (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? Please read the paper! (8) What can you do if you have no crystals?Please read the paper!

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Douglas Instruments Microseeding slide 66 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Please read the paper! (4) How can we avoid salt crystals?Please read the paper! (5) How can we get more diverse crystals?Please read the paper! (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? Please read the paper! (8) What can you do if you have no crystals?Please read the paper!

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Douglas Instruments Microseeding slide 73 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Please read the paper! (4) How can we avoid salt crystals?Please read the paper! (5) How can we get more diverse crystals?Please read the paper! (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? Avoid high salt in your seed stock; (7) Can we harvest seed crystals from microfluidic devices? Please read the paper! (8) What can you do if you have no crystals?Please read the paper!

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Douglas Instruments Microseeding slide 74 Can we predict which solutions the seed crystals will be stable in?

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Douglas Instruments Microseeding slide 76 Appearance of crystals after incubation for one day 1.Wick away the mother liquor 2.Add 10 µl of the solution to be tested 3.Incubate for 5 minutes 4.Wick away the solution added 5.Add another 10 µl of the solution to be tested 6.Incubate overnight 7.Look at the crystals, comparing photos of before and after

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Douglas Instruments Microseeding slide 78 Investigate stability of complex with isothermal calorimetry, fluorescence anisotropy, thermal shift assay etc. Test stability of seed crystals by incubation of uncrushed crystals in the suggested solution for 1 day Try to find a solution that both the seed crystals and the complex are stable in

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Douglas Instruments Microseeding slide 79 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Please read the paper! (4) How can we avoid salt crystals?Please read the paper! (5) How can we get more diverse crystals?Please read the paper! (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? Avoid high salt in your seed stock; remove ingredients.... test by incubation for 1 day (7) Can we harvest seed crystals from microfluidic devices? Please read the paper! (8) What can you do if you have no crystals?Please read the paper!

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Douglas Instruments Microseeding slide 81 Soaking experiments You need a good supply of wells with about 5 crystals per drop Seeding with diluted seed stock is “the only reliable way” to achieve this